Method for analyzing nucleic acids by means of a substrate having a microchannel structure containing immobilized nucleic acid probes

ABSTRACT

A method and apparatus for analyzing nucleic acids includes immobilizing nucleic probes at specific sites within a microchannel structure and moving target nucleic acids into proximity to the probes in order to allow hybridization and fluorescence detection of specific target sequences.

This is a continuation of application Ser. No. 08/848,553, filed Apr.28, 1997, abandoned.

This invention was made with government support under Contract No.DE-AC05-840R21400 awarded by the U.S. Department of Energy to LockheedMartin Energy Systems, Inc. and the government has certain rights inthis invention.

BACKGROUND OF THE INVENTION Field of The Invention

The present invention relates generally to medical and/or biologicaltesting and devices for performing same, and more particularly, to amethod and apparatus for analyzing minute amounts of nucleic acids forthe presence of specific nucleotide sequences. Single-strand DNA probesare bound to specific regions of microchannels in a glass microchipdevice. Sub-microliter volumes of nucleic acid solutions, buffers andother reagents are transported through the channels under electrokineticor hydraulic control. Hybridization of target nucleic acid sequences tocomplementary probes is detected using either fluorescent labels orintercalating fluorescent dyes.

Description of the Related Art

Hybridization analysis is typically performed in microtiter plate wellsor on planar surfaces that contain arrays of DNA probes. Chemicalmanipulations are required to bring about a hybridization test and todetect the results. These manipulations presently include washing ordipping planar arrays into the appropriate chemicals.

The aforementioned procedures suffer from many drawbacks. For example,they are wasteful of expensive reagents and limited sample volumes.Moreover, they are generally not compatible with efficient automationstrategies and thus tend to be time consuming.

A continuing need exists for methods and apparatuses that limit the useof expensive reagents and priceless samples, while simplifying theoverall procedures to require smaller samples and fewer processingsteps.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method and apparatusfor analyzing nucleic acids which simplifies chemical manipulationsrequired to bring about a hybridization test when performing DNAdiagnostics in biomedical, forensic, and research applications.

Another object of the present invention is to provide a method andapparatus for analyzing nucleic acids which minimizes the use ofexpensive reagents and limited sample volumes.

Another object of the present invention is to provide a method andapparatus for analyzing nucleic acids which avoids the necessity ofpre-labeling a target DNA and increases the sensitivity of hybriddetection by reducing background fluorescence due to non-specificsurface adsorption of labeled target DNA.

Still another object of the present invention is to provide a method andapparatus for analyzing nucleic acids which significantly extend theusefulness of hybridization diagnostics by allowing its application tomuch smaller samples and facilitating automated processing.

These and other objects are met by providing an apparatus for analyzingnucleic acids which includes a microchip having a microchannel structureformed therein, at least one portion of the microchannel structurehaving at least one site capable of affixing thereto a probe, and aplurality of reservoirs in communication with the microchannel structurefor introducing at least one of, or a mixture of, a reagent, analytesolution, and buffer.

In another aspect of the invention, a method of analyzing nucleic acidsincludes bonding oligonucleotide probes to a microchannel formed in amicrochip, adding target nucleic acids and fluorescent stains to themicrochannel, and detecting hybridization by fluorescence staining ofdouble-stranded DNA.

These together with other objects and advantages which will besubsequently apparent, reside in the details of construction andoperation as more fully hereinafter described and claimed, withreference being had to the accompanying drawings forming a part hereof,wherein like numerals refer to like elements throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an apparatus for analyzing nucleic acidsaccording to a preferred embodiment of the present invention;

FIGS. 2 and 3 are schematic views of different arrangements of nucleicacid hybridization probes in microchannels;

FIG. 4 is a schematic view of a microchip and microchannel structureaccording to another preferred embodiment of the present invention;

FIG. 5 is a schematic view of a microchip of the present invention;

FIG. 6 is a photomicrograph showing discrimination of target andnon-target DNA at the intersection of microchannels in the inset area ofFIG. 5 after dsDNA staining with fluorescent dye;

FIG. 7 is a schematic view of another apparatus for analyzing nucleicacids according to a preferred embodiment of the present invention; and

FIG. 8 shows fluorescence image profiles of two probe channels afterds-DNA staining with fluorescent dye.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to FIG. 1, a microchip 16 includes a glass substrate 18 and acover plate 20 which covers a microchannel structure 22 formed in theupper surface of the substrate 16. The cover plate 20 is permanentlybonded to the substrate 18. Both the substrate 18 and cover plate 20 arepreferably made of clear glass, and the substrate may preferably be madefrom a standard microscope slide. Alternative construction materialscould include plastics (such as polypropylene, polycarbonate, orpolymethylmethacrylate), silicon, or sapphire.

The microchannel structure 22 is formed using standard photolithographictechniques, and includes a longitudinal microchannel manifold portion24, a first transverse microchannel portion 26 forming an intersection28 with the longitudinal portion 24, and a second transversemicrochannel portion 30 forming an intersection 32 with the longitudinalportion 24.

First and second reservoirs 34 and 36 are in fluid communication withopposite ends of the longitudinal portion 24. The opposite ends act asports to introduce the contents of the reservoirs 34 and 36 into themicrochannel structure 22. Each reservoir can be a cylindrical containeropen at its opposite axial ends, with the ends of the longitudinalportion 24 being in fluid communication with the bottom of thecontainer.

Third and fourth reservoirs 38 and 40 are in fluid communication withopposite ends of the first transverse portion 26. The opposite ends actas ports to introduce the contents of the reservoirs 38 and 40 into themicrochannel structure 22. Each reservoir 38 and 40 is similar inconstruction to the other reservoirs, with the ends of the firsttransverse portion being in fluid communication with the bottom of eachrespective reservoir 38 and 40.

Fifth and sixth reservoirs 42 and 44 are in fluid communication withopposite ends of the second transverse portion 30. The opposite ends actas ports to introduce the contents of the reservoirs 42 and 44 into themicrochannel structure 22. Each reservoir 42 and 44 is similar inconstruction to the other reservoirs, with the ends of the secondtransverse portion being in fluid communication with the bottom of eachrespective reservoir 42 and 44.

One or more types of single-stranded DNA probes 46 are attached atindividual sites within the microchannel portion 24 of the microchannelstructure 22. The design and fabrication of microchips and theelectrokinetic transport of fluids through the microchannels isdescribed in U.S. Ser. No. 08/283,769, filed Aug. 1, 1994, U.S. Pat. No.6,001,229 hereby incorporated by reference. The microchips describedtherein include planar, glass substrates into which the microchannelsare etched photolithographically. The reservoirs typically hold analytesolutions, buffers, reagents, etc. Typical microchannel dimensions are10 μm by 50 μm (depth×width), although channel widths of 1 μm to >100 μmand channel depths of <1 μm to >100 μm may be used. Voltages are appliedto solutions as described in the aforementioned application to produceelectroosmotic flow of fluids or electrophoretic migration of chargedspecies through the channels. Alternatively, pressure (or vacuum) may beapplied to one or more fluid reservoirs to cause reagent flow throughthe channels.

The individual DNA probes may be arranged in a linear pattern, as shownin FIG. 2. An alternative embodiment is shown in FIG. 3, wherein the 46′are arranged in a two-dimensional array in a widened area 48 of thechannel portion 24′. Fluid flow is in the direction indicated by arrows.

Typically, oligonucleotide probes ten to thirty nucleotides long areused for hybridization analysis, although much longer probes, such asDNA restriction fragments or cDNA sequences of >100 nucleotide length,may be used in certain applications.

Oligonucleotide probes may be immobilized by covalent chemical linkageto the surface. In general, such linkage involves derivatization of theglass surface with a silane coupling agent, such as3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. Anoligonucleotide probe bearing an alkylamine group at the 5′ or 3′ endmay then be linked to the surface by direct reaction of its terminalamine with a silane epoxy group or by cross linking the silane andoligonucleotide amines using glutaraldehyde or other amine-reactivebi-functional compounds.

Other immobilization method may also be used. For example,surface-immobilized avidin or streptavidin may be used to bindbiotinylated probes. Non-covalently adsorbed oligonucleotides on glasssurfaces have also been shown to hybridize to target sequences.

In the preferred fabrication method, the probes are attached to the openmicrochip channels and the cover plate is then bound to the substrate bya low temperature technique which does not damage the biomolecules. Sucha low temperature bonding technique is described in application Ser. No.08/645,497, abandoned, entitled “Low Temperature Material BondingTechnique” by J. M. Ramsey, R. S. Foote, and H. Wang, which isincorporated herein by reference. Individual probes may be applied tospecific sites in the channels by micro-pipeting or other means, such asink-jet printing. The separation of individual probes may be facilitatedby preparing the surface with a pattern of reactive, hydrophilic sitesseparated by non-reactive, hydrophobic areas. For example, the glasssurface may be treated with an alkyltrialkoxysilane to produce anon-reactive, hydrophobic surface. Photolithography and chemical etchingor laser ablation may be used to remove the silane layer and expose theglass substrate in a pattern of separated spots. These spots may then betreated with a silane coupling agent as described above to producereactive, hydrophilic spots. An aqueous probe solution applied to anindividual spot would be confined to its hydrophilic site and thusprevented from mixing with different probe solutions in adjacent spots.The intervening hydrophobic regions would also prevent probe mixing inthe case of the other immobilization methods described above.

Alternatively, the probes may be attached to specific sites in thechannels after standard high-temperature cover plate bonding. Threemethods of achieving this are provided as examples:

(1) The functional group of the silane linker (e.g., the amino functionof 3-aminopropylsilane)may be blocked with a photolabile protectivegroup. The silane linkers are then de-protected at specific positions inthe channel by exposure to light through the cover plate using aphotolithographic mask or focused beam. Cross linkers and probes passedthrough the channel would react only at de-protected sites. A series ofseparate de-protection and addition steps are used to attach a number ofdifferent probes to individual sites.

(2) An array of oligonucleotide probes may be photochemicallysynthesized in situ in a parallel fashion.

(3) A channel manifold may be designed to allow the addition of anindividual probe to a given branch or segment of the manifold bycontrolling fluid flows.

In the preferred methodology, nucleic acids, buffers and dyes areelectrokinetically driven through the microchannels containing theimmobilized probes. For example, the following sequence of operationscan be used with the device schematically illustrated in FIG. 4. As seenin FIG. 4, a microchip 50 includes a microchannel structure 52 connectedto a nucleic acid sample reservoir 54, a buffer reservoir 56, a dyereservoir 58, dye buffer reservoir 60, and waste reservoir 62. Ahybridization chamber 64 is disposed in the microchannel structure 52between first and second transverse portions 66, 68 of the microchannelstructure.

A voltage is applied between reservoir 54 which contains the nucleicacid sample being analyzed and reservoir 56 containing nucleic acidbuffer. For buffers containing a high NaCl concentration (desirable forrapid nucleic hybridization) the polarity of reservoir 56 is positiverelative to reservoir 54 and the negatively charged nucleic acidselectrophoretically migrate from reservoir 54 to reservoir 56, passingthrough the hybridization chamber 64. Alternatively, a nucleic acidsolution containing a low salt concentration may be electroosmoticallytransported into the hybridization chamber by applying a positivevoltage at reservoir 54 relative to reservoir 56. Because electroosmoticflow toward reservoir 56 is high relative to electrophoretic migrationtoward the positive electrode, the net movement of nucleic acids will betoward reservoir 56 in the later case. The use of electroosmotic flowversus electrophoretic migration will depend on a number of factors, andmay vary depending on the type of sample being analyzed. The term“electrokinetic transport” includes both electroosmotic flow andelectrophoretic migration.

After the DNA sample reaches equilibrium over the probe sites, thevoltage may be discontinued while hybridization occurs. Adouble-strand-DNA-specific (dsDNA-specific) fluorescent dye is thenelectrokinetically transported through the hybridization chamber 64 byapplying voltages to fluid reservoir 58 which contains a dye andreservoir 60 containing a dye buffer. Because high salt concentrationsare not normally required or desirable for this step, electroosmoticflow is the preferred method of dye addition and the polarity ofreservoir 58 will normally be positive relative to reservoir 60. Severalfluorescent double-strand-specific nucleic acid stains are commerciallyavailable. Many of these stains are positively charged so that theirelectrophoretic migration will be in the same direction as theelectroosmotic flow.

Alternatively, the nucleic acids being analyzed may be pre-labeled withfluorescent groups by well known procedures. Although this later methodcan lead to higher background fluorescence, it may be preferred in caseswhere probes contain self-complementary sequences that can result instable duplex formation and dye binding by the probe itself.

Variations in the chip design and analysis procedure are possible. Forexample, electrokinetically driven washing steps may be included beforeand/or after the dye addition step by applying appropriate voltagesbetween the buffer reservoirs and a waste reservoir 62. Nucleic acid anddye solutions might also be added simultaneously to the hybridizationchamber. As an alternative to electrokinetically driven fluidmanipulation, hydraulic pressure or vacuum may be applied to appropriatereservoirs to control the flow of solutions through the microchannels.

After completion of the hybridization and dsDNA staining steps, if used,the hybridization chamber is examined for the presence of fluorescentlylabeled sites by illumination with exciting light through the coverplate. An epifluorescence microscope and CCD camera may be used, asdescribed below, to obtain a fluorescence image of the entire chamber orportion thereof. Scanning confocal fluorescence microscopy may also beused.

The following examples incorporate the apparatus and methodology of thepresent invention. Each involves the steps of (1) covalently bondingoligonucleotide probes to microchannels, (2) adding target nucleic acidsand fluorescent stains to microchannels by electrokinetic flow, (3)detecting hybridization by fluorescence staining of double-stranded DNA,and (4) discriminating target and non-target nucleic acids.

EXAMPLE 1

A 16-mer oligodeoxynucleotide probe sequence containing a5′-(6-aminohexyl)phosphate [H₂N—CH₂)₆-5′-pCGGCACCGAGTTTAGC-3′] (SEQ IDNO: 1) was covalently attached to the hybridization chamber of aprototype microchip similar to that shown in FIG. 4 by glutaraldehydecross linking with the 3-aminopropylsilane-derivatized glass surface. Acomplementary 16-mer (target sequence) oligodeoxynucleotide in 6×SSCbuffer was then electrophoretically added to the hybridization chamberby applying 0.5 kV between reservoir 56 and reservoir 54 (positiveelectrode at reservoir 54) for thirty minutes. A dsDNA-specificfluorescent dye (TOTO-1, Molecular Probes) in 10 mM Tris-borate buffer,pH 9.2, was then electroosmotically added to the chamber by applying 1.0kV between reservoir 60 and reservoir 58 for 30 minutes. The chip wasexamined by video microscopy using laser excitation (514 nm) offluorescence. Bright fluorescence due to the dsDNA-bound dye wasobserved in the hybridization chamber relative to channels not exposedto the target DNA. The image was recorded on video tape.

In a subsequent similar experiment using the ds-DNA specific dye,PicoGreen (Molecular Probes), quantification by CCD imaging and analysisshowed a 10-fold increase in fluorescence intensity when staining wascarried out after hybridization of the target DNA, relative to theintensity observed by staining prior to the hybridization step.

EXAMPLE 2

The 16-mer oligonucleotide probe of Example 1 was uniformly bound to thechannels of a cross-channel chip shown schematically in FIG. 5 byglutaraldehyde cross-linking. Solutions (50 μM) of the complementary(target sequence) 16-mer oligodeoxynucleotide (T) and anon-complementary (non-target sequence) 16-mer oligodeoxynucleotide (N)in phosphate-buffered saline (PBS) were then added to separate channelsas indicated in FIG. 5, by applying suction at W for 10 minutes. Thechannels were then washed with buffer and dsDNA-specific dye solution(PicoGreen, Molecular Probes) was added to all channels for fiveminutes. The cross-channel intersection was examined by epifluorescencemicroscopy using a mercury lamp illumination source and FITC filters. A1.0 second CCD exposure, shown in FIG. 6 as the insert of the brokenline area of FIG. 5, showed intense fluorescence (dark regions) in thechannel exposed to target DNA relative to that of channels exposed tonon-target DNA or buffer.

In a similar experiment using laser induced fluorescence imaging, asdescribed in application Ser. No. 08/800,241, U.S. Pat. No. 6,056,859,entitled “Method and Apparatus for Staining Immobilized Nucleic Acids”by J. M. Ramsey, R. S. Foote and S.C. Jacobson, incorporated herein byreference, signal intensity from channels exposed to target DNA was10-fold greater than from channels exposed to non-target DNA or buffer.

EXAMPLE 3

Two 16-mer probes [H₂N—(CH₂)₆-5′-GCTAAACTCGGTGCCG-3′ (Probe 1)] (SEQ IDNO: 2) and [H₂N—(CH₂)₆-5′-pCGGCACCGAGTTTAGC-3′ (Probe 2)] (SEQ ID NO: 1)were immobilized in separate channels of a cross-channel chip asindicated in FIG. 7. In FIG. 7, the “T” reservoir is for target DNA, “B”is for PBS buffer and “W” is for waste.

A solution of 16-mer oligonucleotide (50 nM oligonucleotide in PBS)complementary to Probe 1 was induced to flow through both channels for atotal of 15 minutes by applying a vacuum at W. The channels were thenwashed with buffer and treated with a ds-DNA specific dye solution(PicoGreen, Molecular Probes) for two minutes. After washing with 10 mMTris-HCL (pH 8), one mM EDTA (TE) buffer for one minute, the channelswere examined for laser-induced fluorescence using an argon ion laser at488 nm and 100 milliwatts power. Quantitation by CCD imaging, shown inFIG. 8, shows a 4 to 5-fold greater fluorescence in the Probe 1 channelthan in the Probe 2 channel after subtraction of the background signal.

According to the above methods and apparatuses, hybridization analysiscan be performed in a microchip structure that requires lowinstrumentation space and extremely low sample/reagent volumes. Theelectrokinetic transport of samples and reagents facilitates automationof sample/reagent manipulations. Moreover, the detection ofhybridization using double-strand DNA-specific fluorescent dyeseliminates the target DNA labeling step associated with prior arttechniques and increases detection sensitivity.

While the examples referred to above describe nucleic acid probes, themethodology and apparatuses could also be used for other uses including,but not limited to, immobilized antibodies for micro-immunoassays.Numerous biomedical applications can be envisioned.

While the various embodiments have referred to specific reservoirscontaining specific reagents, buffers or samples, mixtures of two ormore substances can be contained in individual reservoirs. For example,a reservoir can contain a mixture of reagent and buffer, buffer andsample, etc.

The many features and advantages of the invention are apparent from thedetailed specification, and thus, it is intended by the appended claimsto cover all such features and advantages of the invention which fallwithin the true spirit and scope of the invention. Further, sincenumerous modifications and variations will readily occur to thoseskilled in the art, it is not desired to limit the invention to theexact construction and operation illustrated and described andaccordingly, all suitable modifications and equivalents may be resortedto, falling within the scope of the invention.

2 1 16 DNA Artificial Sequence Sequence source/note=“syntheticoligonucleotide construct containing a 6-amino hexyl phosphatemodification at the 5′ end” 1 cggcaccgag tttagc 16 2 16 DNA ArtificialSequence Sequence source/note=“synthetic oligonucleotide constructcontaining a 6-amino-hexyl phosphate modification at the 5′ end” 2gctaaactcg gtgccg 16

What is claimed is:
 1. A method for analyzing nucleic acids comprisingthe steps of: a) providing a substrate having a microchannel structurewhich includes at least one microchannel therein; b) immobilizing anumber of different nucleic acid probes within at least a portion ofsaid microchannel structure, at least one said microchannel having aprobe-containing portion with a like number of probe sites, wherein eachof said different nucleic acid probes is immobilized at a discrete probesite; c) moving a target nucleic acid sample under the influence of anelectrokinetic force into the probe-containing portion of saidmicrochannel; d) subjecting said target nucleic acid sample in saidprobe-containing portion of said microchannel to hybridizationconditions; e) labeling with a fluorescent substance one member selectedfrom the group consisting of said target nucleic acid sample and anyhybrids formed in step d; and f) detecting fluorescence emission fromsaid fluorescent substance.
 2. A method according to claim 1, whereinthe step of moving said target nucleic acid sample includeselectroosmotically transporting a solution containing said targetnucleic acid sample through said microchannel structure.
 3. A methodaccording to claim 1, wherein the step of moving said target nucleicacid sample includes electrophoretically migrating said target nucleicacid sample through a solution contained in said microchannel structure.4. A method according to claim 1, wherein said microchannel structure isin fluid communication with at least two fluid reservoirs and the stepof moving said target nucleic acid includes applying electrical voltagesto said at least two fluid reservoirs, one of said reservoirs containinga solution of said target nucleic acid sample.
 5. A method according toclaim 1, wherein any hybrids formed in step d) are labeled with afluorescent double-strand-specific nucleic acid stain in step e).
 6. Amethod according to claim 1, wherein said fluorescent substance iselectrokinetically transported to said probe-containing portion of saidmicrochannel.
 7. A method according to claim 1, additionally comprisingthe step of washing said probe-containing portion of said microchannelbefore step e) to remove unhybridized target nucleic acid sample.
 8. Amethod according to claim 6, additionally comprising the step of washingsaid probe-containing portion of said microchannel after step e) toremove fluorescent substance that is not incorporated into hybridsformed in step d).
 9. A method according to claim 8, wherein saidwashing step comprises electrokinetically transporting buffer throughsaid probe-containing portion of said microchannel probe.